Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

<div><p>RhoH, an atypical small Rho-family GTPase, critically regulates thymocyte differentiation through the coordinated interaction with Lck and Zap70. Therefore, RhoH deficiency causes defective T cell development, leading to a paucity of mature T cells. Since there has been no gain-of-function study on RhoH before, we decided to take a transgenic approach to assess how the overexpression of RhoH affects the development of T cells. Although RhoH transgenic (RhoH^tg) mice expressed three times more RhoH protein than wild-type mice, β-selection, positive, and negative selection in the thymus from RhoH^tg mice were unaltered. However, transgenic introduction of RhoH into Rag2 deficient mice resulted in the generation of CD4⁺CD8⁺ (DP) thymocytes, indicating that overexpression of RhoH could bypass β-selection without TCRβ gene rearrangement. This was confirmed by the in vitro development of DP cells from Rag2^-/-RhoH^tg DN3 cells on TSt-4/Dll-1 stroma in an Lck dependent manner. Collectively, our results indicate that an excess amount of RhoH is able to initiate pre-TCR signaling in the absence of pre-TCR complexes.</p></div

The transgenically expressed HA-tagged RhoH is capable of compensating T cell development in RhoH^-/- mice.

<p>(A) Analysis of RhoH protein expression by western blot in RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes. (B, C) Analysis of RhoH^-/-, RhoH^-/-RhoH^Tg, and RhoH^+/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4^-CD8^- (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4⁺CD8⁺ cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001</p

Analysis of T cell development in RhoH overexpressing mice.

<p>Analysis by flow cytometry of thymocytes and splenocytes from RhoH^+/+RhoH^Tg (red) and RhoH^+/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4^-CD8^-CD44^-CD25⁺ (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH^+/+RhoH^Tg and RhoH^+/+ mice in either MHC^+/+ (C, n = 6) or MHC^-/- (D, n = 5) background. Solid line and dashed line represent RhoH^+/+ and RhoH^+/+RhoH^Tg, respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4⁺ T (F) or CD8⁺ T (G) cells gated on TCRβ⁺ T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.</p

Lck activation is required for RhoH transgene-induced DPs generation without TCRβ-gene rearrangement.

<p>(A) FACS analysis of the <i>in vitro</i> differentiation of thymocytes to the DP stage. DN3 cells from Rag2^-/-, Rag2^-/-RhoH^tg, and Rag2^+/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.</p

The CABIT1 and CABIT2 in Themis have different functions.

<p>(A) CD4 and CD8 profiles of thymocytes and splenocytes of Themis^+/+, Themis^+/+ ΔCore1, and Themis^+/+ ΔCore2 mice. Numbers in plots show the frequency of cells in the indicated area. Bar graphs show absolute number of total thymocytes and thymocyte subsets from Themis^+/+, Themis^+/+ ΔCore1, and Themis^+/+ ΔCore2 mice (mean ± SEM). Data are representative of four independent experiments. (B) Proportion of post-selected CD69⁺ TCR^hi cells in CD4⁺CD8⁺ (DP) thymocytes. Data are representative of four independent experiments. (C) Surface expression of CD25 and CD44 on gated DP thymocytes. Data are representative of four independent experiments. (D) CD44 and CD62L profile of splenic CD4SP and CD8SP cells. Data are representative of four independent experiments. (E) Representative histogram overlays of phosphorylation of ERK. DP thymocytes were stimulated with anti-CD3 plus anti-CD4 Abs for 1min, then intracellular staining of phosphorylated-ERK antibody was performed (solid line). The shaded line is without stimulation. Data are representative of three independent experiments. (F) Absolute number of thymic CD4⁺25⁺Foxp3⁺ Treg cells (mean ± SEM). Data are representative of four independent experiments. Significant differences are noted in the graphs. *p<0.05, **p<0.01. N.S. = not significant.</p

The CABIT1 domain of Themis is important for nuclear localization.

<p>(A) DP-thymocytes from Themis^+/+ mice were fractionated into cytoplasm, membrane and nuclear fractions and immunoblotted with indicated Abs. The CRK and PARP proteins were used as purity controls for the cytoplasmic and nuclear fractions, respectively. Data are representative of more than four independent experiments. (B) Representative micrograph showing localization of Themis (white) in cytoplasm and nucleus stained with DAPI (blue), together with BF (bright field). Data are representative of three independent experiments. (C) Localization of Themis mutants was analyzed by Western blot in cytoplasmic and nuclear extracts. Thymocytes from Themis mutant mice were fractionated into the cytoplasm and nucleus. (D) Bar graph shows that the nuclear/cytoplasmic Themis protein ratio compared with Themis^+/+ mice. Data are representative of more than three independent experiments. Significant differences are noted in the graphs. *p<0.05.</p

Themis mutants lack tyrosine-phosphorylation and Grb2-association.

<p>(A) Analysis of tyrosine-phosphorylation and protein interactions of Themis mutants. Thymocytes from the indicated mutant mice were stimulated with anti-CD3 plus anti-CD4 antibodies. Proteins were immunoprecipitated (IP) with anti-Themis antibody and analyzed by immunoblot (IB) with the indicated antibodies. Data are representative of four independent experiments. (B) Anti-Themis monoclonal antibody (mAb) 2E7 binds to the PRS motif. Immunoprecipitated Themis proteins from Themis^+/+, Themis^−/− ΔCore1, Themis^−/− ΔCore2 and Themis^−/− ΔPRS thymocytes were immunoblotted with anti-Themis mAb 2E7. Data are representative of three independent experiments. (C) Cell lysates from Themis^+/+ thymocytes were sequentially immunoprecipitated with 2E7 and anti-Themis polyclonal antibody (pAb). Grb2 was co-precipitated with anti-Themis pAb but not with 2E7. Data are representative of three independent experiments.</p

Generation of Themis^−/− mice expressing mutant Themis proteins.

<p>(A) Schematic representation of the mutant Themis proteins. (B) Analysis of Themis protein expression by immunoblot using sorted CD69⁻ DP thymocytes from Themis^+/+, Themis^+/−, Themis^−/− and Themis^−/− mice expressing ΔPRS, ΔCore1, ΔCore2, ΔNLS, CAB2-1 mutant, or WT Themis. Data are representative of more than three independent experiments.</p

Expression of the Themis mutants failed to restore positive selection in Themis^−/− mice.

<p>(A) CD4 and CD8 expression profiles of thymocytes or splenocytes from Themis mutant mice. Numbers in plots show the frequency of cells in the indicated area. Proportion (%) of indicated subsets of (B) thymocytes and (C) splenocytes (mean ± SEM). (A-C) Data are representative of more than three independent experiments. Only significant differences against −/− mice are noted in the graphs. *p<0.05, **p<0.01, ***p<0.001.</p

Ribosome accumulation in 5’UTR correlates with translational repression of target genes.

<p>(<b>A-C</b>) Ribosome accumulation in 5’UTRs of ribo-upregulated TKO targets in WT B cells (<b>A</b>), ribo-downregulated TG targets in TG B cells (<b>B</b>), but not in 5’UTRs of other targets (<b>C</b>). Ribosome occupancy in 5’UTR was normalized to the overall ribosome footprint abundance of the same gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.ref096" target="_blank">96</a>]. The first nucleotide of start codon is set as position 0 (grey dashed line). (<b>D</b>) Inverse correlation between ribosome occupancy in 5’UTR and the overall ribosome density on target mRNA in WT B cells. (<b>E</b>) High GC content in 5’UTRs of ribo-upregulated TKO targets.</p